AIM:To develop a new, rapid and accurate reverse dot blot(RDB) method for the detection of intestinal pathogens in fecal samples.METHODS:The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escheric...AIM:To develop a new, rapid and accurate reverse dot blot(RDB) method for the detection of intestinal pathogens in fecal samples.METHODS:The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7, Clostridium botulinum , Bacillus cereus , Clostridium perfringens , Vibrio parahaemolyticus , Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes and Staphylococcus aureus.The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested.Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay.RESULTS:The methods could identify the 12 intestinal pathogens specifi cally, and the detection limit was as low as 103 CFUs.The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%.CONCLUSION:The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.展开更多
Introduction:A quadruplex digital polymerase chain reaction(dPCR)method was developed for the simultaneous detection of Salmonella spp.,Shigella spp.,Vibrio cholerae,and V.parahaemolyticus in wastewater to enhance pat...Introduction:A quadruplex digital polymerase chain reaction(dPCR)method was developed for the simultaneous detection of Salmonella spp.,Shigella spp.,Vibrio cholerae,and V.parahaemolyticus in wastewater to enhance pathogen identification velocity and efficiency.This study established detection limits for these bacterial pathogens and validated the method using environmental wastewater samples.Methods:Specific primers and probes were designed targeting the invA gene of Salmonella,ipaH gene of Shigella,tlh gene of V.parahaemolyticus,and cholera toxin gene ctxA of V.cholerae.The quadruplex dPCR assay underwent rigorous evaluation for analytical sensitivity and specificity.Detection limits were determined using spiked wastewater samples,and the method’s effectiveness was assessed through preliminary testing of 60 environmental wastewater samples.Results:The quadruplex dPCR assay was optimized at an annealing temperature of 58°C.In spiked wastewater samples,the detection limits were 390 CFU/100 mL for Salmonella,11 CFU/100 mL for Shigella,660 CFU/100 mL for V.cholerae,and 640 CFU/100 mL for V.parahaemolyticus.Analysis of 60 municipal wastewater samples revealed pathogen concentrations ranging from 100.9–14,560 copies/100 mL for Shigella,86.5–7,329 copies/100 mL for Salmonella,and 84.5–865.7 copies/100 mL for V.parahaemolyticus.Conclusions:The developed quadruplex dPCR assay demonstrates robust capability for comprehensive surveillance of intestinal bacterial pathogens in wastewater,offering reliable detection even at low concentrations.展开更多
文摘AIM:To develop a new, rapid and accurate reverse dot blot(RDB) method for the detection of intestinal pathogens in fecal samples.METHODS:The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7, Clostridium botulinum , Bacillus cereus , Clostridium perfringens , Vibrio parahaemolyticus , Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes and Staphylococcus aureus.The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested.Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay.RESULTS:The methods could identify the 12 intestinal pathogens specifi cally, and the detection limit was as low as 103 CFUs.The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%.CONCLUSION:The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.
基金Supported by the National Key Research and Development Program(2022YFC3201704-01)from the Ministry of Science and Technology of the People’s Republic of Chinathe Key Project of the Capital’s Funds for Health Improvement and Research,China(2022-1G-4231).
文摘Introduction:A quadruplex digital polymerase chain reaction(dPCR)method was developed for the simultaneous detection of Salmonella spp.,Shigella spp.,Vibrio cholerae,and V.parahaemolyticus in wastewater to enhance pathogen identification velocity and efficiency.This study established detection limits for these bacterial pathogens and validated the method using environmental wastewater samples.Methods:Specific primers and probes were designed targeting the invA gene of Salmonella,ipaH gene of Shigella,tlh gene of V.parahaemolyticus,and cholera toxin gene ctxA of V.cholerae.The quadruplex dPCR assay underwent rigorous evaluation for analytical sensitivity and specificity.Detection limits were determined using spiked wastewater samples,and the method’s effectiveness was assessed through preliminary testing of 60 environmental wastewater samples.Results:The quadruplex dPCR assay was optimized at an annealing temperature of 58°C.In spiked wastewater samples,the detection limits were 390 CFU/100 mL for Salmonella,11 CFU/100 mL for Shigella,660 CFU/100 mL for V.cholerae,and 640 CFU/100 mL for V.parahaemolyticus.Analysis of 60 municipal wastewater samples revealed pathogen concentrations ranging from 100.9–14,560 copies/100 mL for Shigella,86.5–7,329 copies/100 mL for Salmonella,and 84.5–865.7 copies/100 mL for V.parahaemolyticus.Conclusions:The developed quadruplex dPCR assay demonstrates robust capability for comprehensive surveillance of intestinal bacterial pathogens in wastewater,offering reliable detection even at low concentrations.
